Inhibitors of Ras effector signaling are considered the most viable direction for successful development of effective anti-Ras therapies for the treatment of pancreatic ductal adenocarcinoma (PDAC), with most efforts focused on the Raf-MEK-ERK mitogen-activated protein kinase (MAPK) cascade. Eighteen Raf or MEK inhibitors are currently under Phase I-III clinical evaluation. However, signaling reprogramming mechanisms that restore ERK activation downstream of the inhibitor block, or that activate parallel activities to reduce ERK dependency, have severely limited their anti-tumor activities. SCH772984 is a recently developed novel, highly selective ATP-competitive and allosteric ERK1 and ERK2 inhibitor that is currently under Phase Ib clinical evaluation for RAS or BRAF mutant cancers. We propose studies to define signaling mechanisms that overcome ERK inhibition and drive ERK isoform differences, with the long-term goal to advance the clinical development of SCH772984 and other ERK inhibitors. First, our preliminary studies found SCH772984 more effective than MEK inhibition for blocking PDAC cell line anchorage-dependent and -independent growth. However, a subset of PDAC lines showed de novo (primary) resistance to SCH772984. We have also found that high-dose SCH772984 treatment of sensitive PDAC lines resulted in the outgrowth of subpopulations with acquired (secondary) resistance. We will apply druggable genome siRNA screens to identify genes that control de novo versus acquired PDAC resistance to SCH772984. We hypothesize that these studies will identify combination inhibitor approaches that synergistically enhance the anti-tumor activity of ERK inhibitors. Second, surprisingly, despite their high sequence and biochemical identity, we determined that ERK1 and ERK2 display distinct, non-overlapping essential functions in PDAC growth. A genome-wide phosphoproteomics approach will be applied to identify ERK isoform-specific substrates essential for PDAC growth. In addition to delineating novel signaling mechanisms driven by ERK activation, these studies may identify directions for isoform-selective anti-ERK therapeutic strategies. Our application of innovative strategies to study ERK-dependent PDAC growth are high risk; but with the critical importance of ERK in PDAC growth, our findings have high-gain potential for a breakthrough in PDAC therapy.